Objective: The aim of the study was to evaluate the applications of TGF-beta 1 siRNA in cell-seeded scaffold to reconstruct urethra of rabbits. Methods: Autologous oral keratinocytes and TGF-beta 1 siRNA transfected fibroblasts were seeded onto polycaprolactone/silk fibroin/collagen (PSC) electrospun fiber to obtain a tissue-engineeredmucosa. A ventral urethral mucosal defect was created in 16 male rabbits. Then the urethroplasty was performed with autogenic oral keratinocyte- and TGF-beta 1 siRNA transfected fibroblast-seeded PSC electrospun fiber in rabbits of experimental group (n = 8), or with no cells-seeded PSC electrospun fiber in rabbits of control group (n = 8). Retrograde urethrography and histological analyses were performed to evaluate the results of urethroplasty. Results: The oral keratinocytes and TGF-beta 1 siRNA transfected fibroblasts had good biocompatibility with PSC electrospun fiber. After urethroplasty, the urethra was kept in a wide caliber in experimental group, however, the strictures were observed in control group. Stratified epithelial layer and formation of capillary in the epithelial lower layer were observed after 6 months postsurgery in experimental group. Conclusion: Oral keratinocytes and TGF-beta 1 siRNA transfected fibroblasts can be used as a source of seed cells for urethral tissue engineering in rabbits. TGF-beta-silenced fibroblasts are beneficial for the repair of urethral mucosa. PSC electrospun fiber is a good scaffold for tissue-engineering in urethral reconstruction.